Effect of thermal and ensilation treatments on viability of Taenia hydatigena eggs
BY
Birpal Singh Buttar
A dissertation submitted in partial fulfillment of
the requirements for the degree of
DOCTOR OF PHILOSOPHY
WASHINGTON STATE UNIVERSITY
Department of Animal Sciences
May 2010
ii
To the Faculty of Washington State University:
The members of the committee appointed to examine the dissertation of BIRPAL
SINGH BUTTAR find it satisfactory and recommend that it be accepted.
Mark L. Nelson (Co-Chair)
Jan R. Busboom (Co-Chair)
Douglas P. Jasmer
Dale D. Hancock
Douglas B. Walsh
iii
ACKNOWLEDGEMENT
I find writing this section of the dissertation a great opportunity to express
my gratitude towards all those individuals who helped me to finish my graduate
work and this dissertation. I feel my graduate experience, research and
production of this dissertation was a great journey that I will cherish fore ver and
which was not possible without the support of certain individuals.
First and foremost I would like to thank God for giving me the strength,
courage and right directions to get this work accomplished.
I thank my major advisors Dr. Mark Nelson and Dr. Jan Busboom for their
trust in me and their adherent support. Over the past years they have guided me
to develop and understand my scholastic abilities. Their resonating assurance
that “we are in this together” was always inspiring and gave me the coura ge to
walk those few difficult steps that I feared to stumble. I also thank the rest of my
committee Dr. Douglas Jasmer, Dr. Dale Hancock and Dr. Douglas Walsh for their
insightful comments, constructive criticism and for making this research project a
great opportunity for me to learn and evolve as a researcher.
I would like to specially thank Dr. Marshall Lightowlers, University of
Melbourne, Australia for supplying experimental material and consultation on
experimental comport. I admire the selfless support he provided that attests to
his compassion and dedication as a researcher. I would also like to thank Rich
Villa, Dan Snyder, Susan Smart, Angie Mitzel , Nada Cummings, Jeanene de Avila,
iv
John Lagerquist and Ting Jiang for all the support they provided t o make this
research seem so easy for me. Also, I am grateful to Matrix and Amigo, my two
research dogs, for making this research possible.
Finally, I would like to thank my family: Harsimran, Sukhreet, Sonia and my
parents for their love and support. This work would not have been possible
without their help.
Birpal S. Buttar
v
Effect of thermal and ensilation treatments on viability of Taenia hydatigena eggs
ABSTRACT
BY
Birpal Singh Buttar, Ph.D.
Washington State University
May 2010
Co-Chairs: Mark Loge Nelson, Jan Roger Busboom
In the Pacific Northwest USA feeding of potato co–products has been speculated
to result in greater prevalence of beef cysticercosis caused by Taenia saginata as
compared to rest of the USA. A Taenia hydatigena model was used to assess the effect of
heat and ensilation treatments on viabilities of eggs. The T. hydatigena life cycle was
maintained under laboratory conditions by passing the parasite through a canine–ovine
cycle. For studying effect of heat, in vitro and in vivo experiments were carried out at
temperatures ranging from room temperature (22°C) to 60°C for five minutes each. To
study the effect of ensilation, in vivo study was conducted to analyze the effect of 0, 7,
14, 21 and 28 days of ensilation of minced potato on viability of T. hydatigena eggs.
Effect of in vitro heat treatment was analyzed using sigmoidal four–parameter model
and resulted in 99.47% reduction in viability at 60.00°C. In vivo heat treatments caused
linear decrease in viability at the rate of 0.32% per degree Celsius with 100% reduction
occurring at 57.38°C. After ensilation, maximum reduction in viability of 0.10±3.72%
was attained after 18.59±3.65 days of ensilation. Similar heat and ensilation treatments
or a combination of the two may also be effective against T. saginata and may help to
reduce occurrence of beef cysticercosis.
vi
TABLE OF CONTENTS
Acknowledgement .......................................................................................................................... iii
Abstract ........................................................................................................................................... v
List of tables ................................................................................................................................... viii
List of figures...................................................................................................................................... x
Chapter 1 Review of literature .................................................................................................... 1
1.1 Introduction ..................................................................................................................................................2
1.2 Biology .............................................................................................................................................................3
1.3 Life cycle .........................................................................................................................................................4
1.3.1 Eggs ..........................................................................................................................................................4
1.3.2 Cysticerci ...............................................................................................................................................6
1.3.3 Adult tapeworm .................................................................................................................................7
1.4 Epidemiology ................................................................................................................................................8
1.4.1 Role of eggs...........................................................................................................................................8
1.4.2 Role of cysticerci ............................................................................................................................. 10
1.4.3 Role of adult tapeworm ............................................................................................................... 11
1.5 Economic Impact ..................................................................................................................................... 12
1.6 Present control strategies and their short comings ............................................................... 12
1.6.1 Preventing cattle from developing infective stages....................................................... 12
1.6.2 Preventing human infection ...................................................................................................... 13
1.6.3 Preventing egg dispersal ............................................................................................................ 15
1.6.4 Treating feedstuffs to prevent infection of beef animals ............................................ 16
1.7 Studying Taenia spp. viability of eggs............................................................................................ 18
1.7.1 Hatching and activation .............................................................................................................. 18
1.7.2 Staining ................................................................................................................................................ 19
1.7.3 In vitro culture ................................................................................................................................. 20
1.7.4 In vivo and surrogate models .................................................................................................... 20
vii
1.8 Objectives .................................................................................................................................................... 21
1.9 References ................................................................................................................................................... 23
Chapter 2 Thermal killing of Taenia hydatigena eggs ........................................................ 36
2.1 Introduction ............................................................................................................................................... 37
2.2 Materials and methods ......................................................................................................................... 40
2.2.1 Taenia hydatigena eggs ............................................................................................................... 40
2.2.2 In vitro experiment ........................................................................................................................ 42
2.2.3 In vivo experiment ......................................................................................................................... 44
2.2.4 Statistical analysis .......................................................................................................................... 45
2.3 Results ........................................................................................................................................................... 47
2.3.1 In vitro results .................................................................................................................................. 47
2.3.2 In vivo results ................................................................................................................................... 47
2.4 Discussion ................................................................................................................................................... 49
2.5 References ................................................................................................................................................... 54
Chapter 3 Effect of ensilation of potato on viability of Taenia hydatigena eggs .......... 66
3.1 Introduction ............................................................................................................................................... 67
3.2 Materials and methods ......................................................................................................................... 70
3.2.1 Eggs ....................................................................................................................................................... 70
3.2.2 Ensilation............................................................................................................................................ 70
3.2.3 Lambs ................................................................................................................................................... 71
3.2.4 Statistical analysis .......................................................................................................................... 72
3.3 Results ........................................................................................................................................................... 73
3.4 Discussion ................................................................................................................................................... 74
3.5 References ................................................................................................................................................... 77
Chapter 4 Conclusion ................................................................................................................... 83
viii
LIST OF TABLES
Table 1.1 Taxonomy of tapeworms of genus Taenia. .............................................. 32
Table 1.2 Reported bovine cysticercosis outbreaks in North America. .............. 33
Table 1.3 Estimates of economic losses per animal due to cysticercosis. .......... 34
Table 2.1 In vitro effect of five minutes of thermal treatment of T.
hydatigena eggs on percent recovery of oncospheres and
percent activation ex–shelled with 1% sodium hypochlorite
and activated with 50 % bile treatments. ............................................... 59
Table 2.2 Sigmoidal four–parameter model parameter estimates for in
vitro percent activation of ex–shelled T. hydatigena eggs in
response to heat treatment for five minutes at various
temperatures. ................................................................................................ 60
Table 2.3 Average number of calcified and non-calcified cysticerci
recovered from lambs infected with T. hydatigena eggs heat
treated for five minutes at 22 (control), 50, 60°C. ................................ 61
Table 2.4 Average weight (±SE) of lambs infected with heat treated T.
hydatigena eggs at different temperatures. The effect of heat
treatment on weight gains was not significant. ..................................... 62
ix
Table 3.1 Average number of calcified and non-calcified cysticerci
recovered from lambs infected with T. hydatigena eggs ensiled
in minced potato for different time periods. .......................................... 80
Table 3.2 Average weight (±SE) of lambs infected with T. hydatigena
eggs ensiled for various time periods. Average daily weight
gains of lambs did not vary significantly with length of
ensilation (α=0.05). ..................................................................................... 81
x
LIST OF FIGURES
Figure 1.1 Critical control points in life cycle of Taenia saginata . ........................ 35
Figure 2.1 Sigmoidal four–parameter model for observed In vitro percent
activation of T. hydatigena oncospheres after heat treatment
for five minutes at various temperatures (R2 = 0.8718). ..................... 63
Figure 2.2 Comparison of liver surfaces of lambs infected with T.
hydatigena eggs heat treated at (a) 22 (control), (b) 50 and (c)
60°C. ................................................................................................................ 64
Figure 2.3. Linear regression response of percent recovery of T.
hydatigena cysticerci recovered as a percent of heat treated
eggs administered at various temperatures. (Y= –0. 318 X +
18.249, R² = 0.8767) .................................................................................... 65
Figure 3.1 In vivo response of percent recovery of T. hydatigena cysticerci
recovered as a percent eggs administered after ensilation in
minced potato for 0, 7, 14, 21 and 28 days. The data points are
average for the treatment with standard error bars. The
regression equation was Y= –1.00 X + 18.69 with plateau
starting at 18.59±3.65 days. ...................................................................... 82
1
Chapter 1 REVIEW OF LITERATURE
2
1.1 INTRODUCTION
Taenia saginata, T. asiatica and T. solium are three major meat borne
zoonotic tapeworms. They cause taeniosis in humans and cysticercosis in meat
animals (beef cattle and swine), respectively. Due to zoonotic importance, these
parasites detrimentally impact the meat industry. Characteristics like high
fecundity, resistant life stages, and co–existence with its hosts make them highly
adaptable to their complex two–host life cycle. Also, due to these characteristics,
these tapeworms are prevalent globally.
Taenia saginata (beef tapeworm) has its major impact on the beef industry.
While T. saginata is highly prevalent in Latin America, Africa, Asia and some
Mediterranean countries, it is also found in other regions with low levels of
prevalence (Murrell and Dorny, 2005). Based on unpublished 2009 United States
Department of Agriculture (USDA) meat inspection data, the prevalence of bovine
cysticercosis in USA and the Northwest USA was 0.003% and 0.052%,
respectively. Based on USDA meat inspection data the prevalence of cysticercosis
in the Northwest has been the highest in the USA at least since 1984. The
relatively high prevalence in the Pacific Northwest (PNW) results in increased
economic losses to the feedlot industry in the region. Potato co–products
contaminated with T. saginata eggs are considered the most likely source of
infection leading to higher prevalence of cysticercosis in the PNW cattle (Hancock
et al., 1989; Yoder et al., 1994). This relationship provides the primary topic of
research that will be described here.
3
1.2 BIOLOGY
Taenia saginata is closely related to and has evolved from tapeworms of
wild canids. Predecessors of T. saginata evolved about 2.0–2.5 million years ago
to include humans as the definitive host when ancestors of Homo sapiens adopted
omnivorous diet to include bovid preys in diet; and later this relation was
intensified during domestication of bovids about 10,000 years ago (Hoberg et al.,
2000; Hoberg et al., 2001; Hoberg, 2002, 2006) . T. saginata has evolved to have
humans as their only definitive host. Based on phylogenetic cladogram, T.
hydatigena , a canine tapeworm, is closely related to T. saginata (Hoberg et al.,
2001) and may be useful as a surrogate organism to study characteristics of T.
saginata.
Parasitic tapeworms of genus Taenia are long, segmented worms with two
host life cycle. Body length may vary from less than 10 cm to more than 1 ,000 cm
(Hoberg et al., 2000). The body of an adult tapeworm is divided into distinct
scolex (head), neck (unsegmented body) and strobila (segmented body).
Taxonomic classification of tapeworms in the genus Taenia is shown Table 1.1.
Previously two distinct life stages of the parasite, adult and metacestode were
considered different species. The morphologically dissimilar life stages and were
wrongly assigned to two different genera, Taenia and Cysticercus . Intermediate
life stage of T. saginata, T. solium , T. hydatigena, T. ovis , T. taeniaeformis were
named C. bovis, C. cellulose , C. tenuicollis , C. ovis and C. fasciolaris, respectively
(Soulsby, 1982 p.107-116; Arme and Pappas, 1983). For a recently identified
4
asian tapeworm some confusion exists for an exact scientific name and has been
referred to as T. saginata asiatica (Flisser et al., 2004; Scandrett, 2007) or T.
asiatica (Hoberg, 2006; Youn, 2009).
1.3 LIFE CYCLE
Epidemiology, disease and control strategies related to Taenia saginata are
entertwined with the life cycle of the parasite. Life cycle of Taenia saginata
involves intermediate (cattle) and definitive (Humans) host species; and three
distinct stages – eggs in environment, cysticerci in the cardiac and skeletal
muscles of the intermediate host and adult tapeworms in the small intestine of
definitive host (Figure 1.1).
1.3.1 EGGS
Infected humans pass terminal mature segments known as gravid
proglottids that contain infective eggs. These proglottids are 6 –22 mm in length
and 6.5–9.5 mm in width (Hoberg et al., 2000; Flisser et al., 2004; Murrell and
Dorny, 2005) and are released in to the intestinal lumen. In T. saginata infections
about 10 gravid proglottids are released from humans per day and each
proglottids contains 50,000–80,000 eggs (Soulsby, 1982; Murrell and Dorny,
2005). A single adult worm can shed 10.7 billion eggs over an average life span of
18 years (Schapiro, 1937). The proglottids of T. saginata are motile and can
actively crawl out of the anus or be shed with feces (Soulsby, 1982). Once in the
environment the eggs can survive for variable time periods depending on the
5
environmental factors. Eggs of T. saginata are infective to ruminant intermediate
hosts, especially cattle, leading to bovine cysticercosis.
Taenia saginata eggs are oval brown structure and 46–50 µm by 39–41 µm in
diameter, made up of a series of outer coverings that bound the hexacanth (six –
hooked) tapeworm larva known as oncosphere (Nieland, 1968; Soulsby, 1982 p.
108; Jabbar et al., 2009). The different layers of the egg are shed in three stages.
The outer most layer consisting of shell/capsule and outer envelope is fragile and
is lost during escape of the egg from the uterus within a gravid proglottid. T he
second layer is made up of keratin blocks and is known as embryophore. The
embryophore is thick and is visible as a radiating brown layer with radial
striations. Eggs in the environment retain this layer. Once ingested by the bovine
intermediate host, the embryophore is lost by the action of digestive juices in
stomach of vertebrate intermediate host. Under laboratory conditions, sodium
hypochlorite may be used to remove this layer (Laws, 1967; Wang et al., 1997).
The third and the inner most layer is the oncospheral membrane made up of a
pair of laminae with closely spaced vesicles (Nieland, 1968). The oncospheral
membrane is lost by the process of activation after exposure to bile in the
duodenal region of the intermediate host (Soulsby, 1982 p.110; Smyth and
McManus, 1989 p.193; Singh and Prabhakar, 2002) . Under laboratory conditions,
frozen or fresh bile may be used for activation of the oncospheres. Species
specificity of Taenia spp. for intermediate hosts is attributed to properties of bile
although no exact components of bile responsible are known (Smyth and
6
McManus, 1989 p.193). Activation is indicated by active movement of hooks. The
three pairs of keratinized hooks develop from oncoblasts (hook forming cells)
and are attached to 18 cell hook muscle system (Jabbar et al., 2009).
The larval stage of Taenia spp. is not as host specific as the adult stage is.
While cattle are the most important species, other ruminants – llama, reindeer,
sheep, goat, roe deer, fallow deer and lagomorphs may also act as intermediate
hosts (Soulsby, 1982 p.108; Cabaret et al., 2002) . Cattle are infected when they
ingest feed or water contaminated with eggs or gravid proglottids from human
sources. In cattle, the eggs hatch to release the oncospheres which further
develop into cysticerci in striated and cardiac muscles.
1.3.2 CYSTICERCI
After activation the T. saginata oncospheres penetrate through the
intestinal mucosa to reach the general circulat ion. The oncospheres then reach
various tissues. T. saginata has preference for striated, cardiac and smooth
muscles especially the diaphragm, tongue, esophagus, masseter, triceps, thigh and
heart but may be found elsewhere (Soulsby, 1982; Kebede et al ., 2008). Based on
postmortem examination of 42 beef calves, Scandrett (2009) concluded that
among the traditional sites, heart was the most and esophagus was the least
reliable site for localization of T. saginata cysticerci among the sites listed.
In transition from multicellular oncospheres to fluid filled cysticerci the
hooks are lost; there is a rapid increase in surface area with development of
7
surface microvilli and generation of a strong antigenic response by the host
(Engelkirk and Williams, 1982; Jabbar et al. , 2009). In muscle tissue, oncospheres
develop into encysted cysticerci stage of T. saginata (Abuseir et al., 2006). Earlier
these cysticerci were thought to be caused by a distinct species, independent of T.
saginata and was named Cysticercus bovis (Kassai, 2001). The lesions caused by
these cysticerci usually cause no any clinical disease or may cause mild clinical
signs in heavy infections, which includes pyrexia, anorexia, muscular weakness
and emaciation (Arme and Pappas, 1983 p. 519; Radostits et al., 2000 p. 1386) .
The infective stage (mature cysticercus) is reached in ten weeks and can remain
viable for up to nine months or more. Cysticerci that die at predilection sites are
calcified by the host defense system and are easily detectable post –mortem.
1.3.3 ADULT TAPEWORM
Humans usually become infected by consuming cysticerci along with
“measley beef” (raw or undercooked infected meat). In the human stomach, the
wall of the cysticerci is digested and young tapeworm is released. The parasite
reaches the small intestine and attaches to the wall with sucker disks p resent on
the scolex (head). The adult tape worm develops in three months and may grow
to 4 to 12m in length (Murrell and Dorny, 2005 p.4). Unlike T. solium , a rostellum
and hooks are missing from the scolex of T. saginata, which provides a diagnostic
feature to differentiate these two tapeworms of humans. The adult tapeworms are
hermaphroditic and following reproduction, eggs develop in gravid proglottids
that are released into the environment.
8
1.4 EPIDEMIOLOGY
All three life stages of T. saginata viz. eggs in environment, cysticerci in
beef cattle and adult tapeworms in humans play important roles in the
epidemiology of the parasite (Murrell and Dorny, 2005). In recent times,
intensification of the beef industry and migration of humans have also played
important epidemiological role.
1.4.1 ROLE OF EGGS
In regions with high prevalence of taeniosis, the possibility of
environmental contamination with T. saginata eggs is really high due to release of
up to 800,000 eggs by each adult worm per day. Subsequently, high resistance to
environmental deterioration enables these eggs to survive for a long time and
possibly relocate to distant locations via different means. Once passed by humans,
eggs can get access to feedstuffs through sewage, water contamination, poor
hygienic practices, or mechanical carriers (humans, birds, arthropods,
earthworms) (Slonka et al., 1975; Slonka et al., 1978; Fertig and Dorn, 1985;
Cabaret et al., 2002; Murrell and Dorny, 2005) .
In the environment, eggs may survive for different lengths of time mainly
depending upon temperature and moisture conditions. The best survival rates are
at low to moderate temperatures and high moisture condit ions. The eggs viability
is quite variable depending upon the prevailing environmental conditions. Even
under similar conditions the survivability of Taenia spp. eggs has been reported
to be quite variable (Froyd, 1962). Eggs remain viable for 11 weeks in water at
9
24°F (-4.44°C) under laboratory conditions (Lucker, 1960), 413 days on pastures
in Kenya (Duthy and Someren, 1948), 60-80 days in grass silage at 10°C in
Germany (Enigk et al., 1969) and 21 days in stored dry hay (Lucker and Douvres,
1960; Murrell and Dorny, 2005).
Fields fertilized with sewage contaminated with T. saginata eggs have been
reported to be an important source of infection for feedlot animals (Cabaret et al.,
2002). The eggs are able to survive all forms of sewage and sludge treatments
(Cabaret et al., 2002; Murrell and Dorny, 2005) . Apart from use as a fertilizer,
municipal sewage may get access to pastures by leakage or flood (Fertig and
Dorn, 1985), or may contaminate the water source of the feedlot (Lees et al.,
2002; Scandrett and Gajadhar, 2004) . Among sludge treatments, lagooning of
sludge at 7°C for 28 days has been reported to be the most successful treatment
causing more than a 99% reduction in viability of T. saginata eggs (Bruce et al.,
1990).
In the Northwest USA, potato co–products are considered the source of
infection of T. saginata eggs to beef cattle at feedlots (Hancock et al., 1989; Yoder
et al., 1994; Nelson, 2003). Potato processing plants in the area generate low cost
co–products in high volumes for the feedlots (Bradshaw et al., 2002; Nelson,
2003). Potato co–products are usually stored in pits at the feedlot for a short time
and are fed to feedlot cattle without processing. Potato co –products may get
contaminated at potato processing plants or at feedlots through sewage water or
infected workers. Since the proglottids are motile and can actively migrate out of
10
infected humans, contamination may not always require direct contact with
human feces. Low to moderate temperature conditions in the region and high
moisture contents of the potato co–products may prolong the survival of T.
saginata eggs.
1.4.2 ROLE OF CYSTICERCI
Since there are no clinical signs associated with bovine cysticercosis in live
animals, the disease is diagnosed postmortem in the form of cysticerci found at
time of routine meat inspection. Based on historic data, both endemic and
epidemic occurrences of beef cysticercosis are possi ble. Epidemics usually
originate from a point source of infection that infects many animals at the same
time. Various sources of infection for cattle may be eggs in silage, cattle pens,
feed bins, pastures, and/or water supplies that have been contaminated with
infected human feces, sewage, or sewage effluent (Weedon, 1987). Outbreaks of
bovine cysticercosis have been reported in the North America (Table 1.2). These
outbreaks were reported mostly based on postmortem inspections. Meat
inspectors may fully condemn the affected carcass or condemn affected parts
depending upon severity of the condition for public health concerns.
Condemnation adds to economic losses and leads to heavy unexpected losses to
the beef industry.
In 2009, the Northwest contributed 4% of the USA beef slaughter but 85%
of the bovine cysticercosis measles cases identified by USDA inspection. The
prevalence was 0.052% in the Pacific Northwest compared to 0.003% in the USA.
11
1.4.3 ROLE OF ADULT TAPEWORM
Globally, the prevalence of T. saginata infections in humans may be divided
into three regions. A highly endemic (>10%) region in central and east Africa, a
moderately endemic (0.1–10%) region in Europe, SE Asia and South America and
a low endemic (<0.1%) region in USA, Canada and Australia (Murrell and Dorny,
2005). Like bovine cysticercosis, taeniosis in many humans may occur from a
point source of infection but there are seldom any reports of epidemics of T.
saginata taeniosis. Most infected humans are clinically normal but shed gravid
proglottids in feces for years (Garcia et al., 2003b). This situation of the disease
makes it impossible to prevent cysticercosis outbreaks.
Taeniosis in USA has not been studied extensively and is poorly understood
(DeGiorgio et al., 2005). In addition to beef as source of infection, immigrants
from endemically infected countries are also a cause of increased cases of bovine
cysticercosis and taeniosis in USA (Flisser et al., 2004; DeGiorgio et al., 2005;
Macpherson, 2005). Unlike bovine cysticercosis, there are no reports of outbreaks
of taeniosis in humans in USA because the infection in most cases is sub –clinical
and has a chronic course of infection.
With advancement in knowledge of public health, stricter post mortem
regulations have made detection and diagnosis of bovine cysticercosis a standard
postmortem practice. If lesions are detected , carcasses are condemned or
appropriately treated to prevent transmission of the disease to humans. But this
12
does not prevent the economical losses to the beef industry. Thus, now the aim of
the investigators is to prevent the exposure of feedlot cattle to T. saginata eggs.
1.5 ECONOMIC IMPACT
Cysticercosis may lead to sever losses to feedlots because of complete
condemnation or carcass processing. Table 1.3 shows the estimates of loss per
animal due to cysticercosis. These losses have risen 369% in 21 years and thus ,
on an average, 18% per year.
1.6 PRESENT CONTROL STRATEGIES AND THEIR SHORT COMINGS
Potential control strategies may be described as four critical control points
in the life cycle of T. saginata (Figure 1.1). These critical control points are (A)
Preventing cattle from developing infective stages; (B) Preventing human
infection; (C) Preventing eggs’ dispersal; and (D) Treating feedstuffs to prevent
infection of beef animals.
1.6.1 PREVENTING CATTLE FROM DEVELOPING INFECTIVE STAGES
This may be achieved by vaccinating or treating animals. Reports of control
with vaccination may be a promising control strategy but treatment of infected
animals is not possible in large scale production systems since cysticercosis is
asymptomatic and the disease cannot be diagnosed in live animals. Substantial
work has been done on vaccine development recently. Recombinant vaccine
having two antigens TSA–9 and TSA–18 confer 99.8% protection against T.
saginata eggs in cattle (Lightowlers, 1996; Lightowlers et al., 1996) .
13
While the vaccines against Taenia spp. have been reported to be efficacious,
it may not be a feasible solution in USA due to various reasons. USA has
historically a low prevalence of T. saginata that makes vaccination not very cost
effective. For instance, the highest prevalence of cystic ercosis in the USA in 2006
was 0.067% in the Northwest USA; that is every 1 out of 1493 was infected. Thus
to protect a single animal in the northwest, vaccination of 1493 animals would be
required. If the vaccine costs $1 per dose, the cost of $1493 would be required to
effectively protect a single infected animal which is more than actual present loss
per animal ($1186) due to cysticercosis. Economics will discourage production of
a commercial vaccine since the demand will be low.
Since the disease involves two hosts, vaccinating cattle alone will never
eliminate cysticercosis till human sources that pass eggs are controlled. Further,
even if the human population in the area is diagnosed and treated, it may not be
possible to stop immigrants from bringing in new T. saginata infections to the
region.
1.6.2 PREVENTING HUMAN INFECTION
The most effective method of killing T. saginata cysticerci from meat is
cooking to minimum of 71°C (USDA, 2001). Due to personal preferences, cultural
or religious practices, proper cooking to recommended temperatures may not be
always achieved in human diets. This may expose individuals to taeniosis. To
address this, better public health practices have been adopted that has led to the
adoption of stricter postmortem inspection techniques against cysticercosis.
14
To date postmortem meat inspection is the most relied upon method of
controlling T. saginata . Visual diagnosis is performed postmortem by an
authorized meat inspector by dissecting predilection muscle sites. T. saginata
cysticercosis is usually diagnosed as calcified or rarely as non calcified cyst icerci
at predilection sites. A heavily infected carcass is totally condemned. In the USA,
Food Safety and Inspection Service (FSIS) directive requires post –mortem
inspectors to inspect heart, tongue, esophagus and other muscles for live, dead or
degenerated Taenia spp. cysticerci (FSIS, 2007). Detection of even a single live,
dead or degenerated cyst from a carcass or iginating from a producer, warrants a
detailed inspection of all other carcasses originating from that source and to
notify the health department of the state and veterinarian in –charge at Animal
and Plant Health Inspection Service (APHIS) of USDA. Dependi ng on the extent of
infection, the carcass may be fully condemned or held for refrigeration or heat
treatment. Mildly infected carcass or boned meat needs to be refrigerated at 15°F
(–9.44°C) or less for 10 or 20 days respectively. Throughout heat treatmen t at
140°F (60°C) of the mildly infected carcass is also acceptable.
Despite rigorous inspection directives, postmortem inspection is not fool
proof. Diagnosis of cysticercosis has been reported to be 3 –10 times less
efficacious depending upon meat inspec tor’s motivation, and experience; and the
extent and stage of infection in a particular carcass (Abuseir et al., 2006).
Moreover, even if postmortem inspection was fully efficacious, it will not preven t
15
economic loses to commercial beef producers and feeders due to total
condemnation, freezing or heating costs.
1.6.3 PREVENTING EGG DISPERSAL
Egg dispersal may be controlled by treating humans, ensuring high
standards of personal hygiene or by ensuring that all mechanisms by which egg
transmission from humans to beef are blocked to prevent egg dispersal. Human
anthelmintic treatment and personal hygiene are particularly important with
those in close contact with beef animals. Humans may be effectively treated with
common anthelmintics like praziquantel, niclosamide, buclosamide or
mebendazole (Garcia et al., 2003a). If diagnosed, the treatment is quite
efficacious but the diagnosis of infected humans is difficult. This is because there
are almost no clinical signs associated with taeniosis and even if a stool sample is
submitted, the sensitivity of stool test is low (Garcia et al., 2003b).
Sewage water application to feedstuffs, water contaminated with infected
human feces and direct transfer from infected farm workers are major channels of
dispersal of T. saginata eggs to beef animals while birds, arthropods and
earthworms form a minor channel of transmission (Slonka et al., 1975; Slonka et
al., 1978; Fertig and Dorn, 1985; Cabaret et al., 2002; Murrell and Dorny, 2005) .
Present sewage treatments do not effectively kill Taenia spp. eggs (Bruce et al.,
1990; Cabaret et al., 2002; Murrell and Dorny, 2005).
16
1.6.4 TREATING FEEDSTUFFS TO PREVENT INFECTION OF BEEF ANIMALS
Since all other control strategies fail to effectively control transmission of
eggs to feedstuffs, this control strategy may be implemented by treating
feedstuffs before feeding them to animals. Since heat and lack of moisture kills
the eggs, feeds may be pelleted to effectively eliminate chances of infection. But
some feedstuffs that may harbor Tania may not be easy to treat like potato co–
products in the Northwest USA. It may not be possible to remove bound water
from potato without altering its nutritive value. Excessive heating has been tried
by some beef operations in the region but this has gelatinized the potato starch
leading to rumen acidosis (Nelson, 2003). A possible control strategy under this
situation may be heating the feedstuff to an optimum time temperature
combination that will not gelatinize potato co –product. Alternatively, the potato
co–products may be ensiled before feeding. The author is not aware of any
research studies that have made these measurements.
1.6.4.1 Heat treatment
In a normal life cycle, Taenia spp. eggs undergo a sudden change in
temperature when they are ingested by vertebrate intermediate host. The eggs
are able to survive this change due to presence of heat shock proteins (Benitez et
al., 1998; Vargas-Parada et al., 2001; Ferrer et al., 2005) . Varagas-Parada et al.
(2001) reported that metacestodes of T. solium and T. crassiceps responded to
incubation at different temperatures by increasing production of heat shock
proteins and this response was lost after heat treatment at 46°C for 30 minutes
17
and was thus lethal. Similar or higher temperatures may or may not be lethal to
Taenia spp. eggs and the author is not aware of any study of the effects of heat on
viability of eggs of Taenia spp. Williams and Colli (1970) studied effect s of heat
on activation rates of T. hydatigena eggs and reported highly variable results,
owing to unknown factors, but did conclude that no activation was observed after
5 minutes at 55°C, 2 minutes at 60°C and 1 minute at 65°C. They also tried an in
vivo model by intraperitoneal inoculation of heat treated ex–shelled and activated
oncospheres into jirds (Meriones unguiculatus) but concluded the system was
unreliable. One of the effects of temperatures from 7 to 38°C was to promote
aging of mature and maturation of immature Taenia spp. eggs (Gemmell, 1977).
While heat treating potato co–products to eliminate the T. saginata threat,
it is important not to cause potato gelatinization. Complete potato gelatinization
required 30–60 minutes of heat treatment at 64.2°C (Shiotsubo, 1983, 1984).
Further, 60 minutes of in vitro heat treatment of potato at 55, 57.5, 60, 62.5 and
65°C results in 0, 46, 73, 88 and 100% gelatinization, respectively (Parada and
Aguilera, 2009).
1.6.4.2 Ensilation
Personal communication with Washington State Cattle Feeders suggests that
feeding fresh potato co–products has led to increased cysticercosis cases in past.
Potato co–products have been ensiled for at least 21 days in different
experiments at Washington State University and no case of cysticercosis has ever
been reported despite higher prevalence of cysticercosis at other feedlot
18
operations in the region that use similar potato co –products but did not ensile for
that long (Nelson, 2009). Lagooning of sludge at 7°C for 28 days causes more than
99% reduction in viability of T. saginata eggs (Bruce et al., 1990). Ensilation of
potato under similar conditions may yield similar results.
1.7 STUDYING TAENIA SPP. VIABILITY OF EGGS
Understanding behavior, ultrastructure and mortality characteristics of
Taenia spp. eggs is critical to devise techniques to control rates of cysticercosis in
intermediate hosts. Owing to complex structure of Taenia spp. eggs and lack of
complete knowledge of structural and behavioral properties, accessing viability
of Taenia spp. eggs still poses a significant challenge. Different in vivo and in vitro
techniques used to assess viability of Taenia spp. eggs include staining, activation
analysis, culture, infection of intermediate hosts and lab animals (Williams and
Colli, 1970; Wang et al., 1997; Minozzo et al., 2002; Chapalamadugu, 2006;
Kyngdon et al., 2006; Chapalamadugu et al., 2008).
1.7.1 HATCHING AND ACTIVATION
Hatching and activations of Taenia spp. eggs are two important processes
that occur under natural conditions that release active oncospheres in the
intermediate host. These two processes may be simulated u nder in vitro
conditions to isolate oncospheres from two important barriers – embryophore
and oncospheral membrane. Hatching refers to removal of the thick brown
embryophore of Taenia spp. egg caused by gastric juices and activation refers to
active movements of the oncosphere leading in escape from the oncospheral
19
membrane by the action of bile. The methodology for in vitro hatching and
activation has improved over time. Silverman (1954) initially developed hatching
technique and used gastric and intestinal enzymatic preparations but the
technique resulted in inconsistent results by many other investigators. Laws
(1967) described the use of sodium hypochlorite to effectively hatch T.
hydatigena , T. ovis, T. pisiformis and Echinococcus granulosus eggs. Exposure of
Taenia spp. eggs to 2% sodium hypochlorite for 10–30 minutes did not cause any
reduction in viability based on activation and culture results (Osborn et al.,
1982). Use of sodium hypochlorite for hatching has been widely adopted by
investigators as a replacement for gastric enzymes (Lightowlers et al., 1984;
Wang et al., 1997; Chapalamadugu et al., 2008). To enhance activation rates,
various other substances like carbon dioxide and sodium hypochlorite have been
recommended but their use have not been widely accepted and sometimes
discouraged. Activation is still a poorly understood process that yields quite
variable results with different batches of eggs of same species. A part ial reason
for this variability has been attributed to difference in ma turation levels of
Taenia spp. eggs, even within a single proglottid. Also, different eggs may require
different lengths of incubation before they exhibit activation.
1.7.2 STAINING
For vital staining with methylene blue, neutral red , or Janus B green,
hatching and activation are required as prerequisite s (Smyth and McManus, 1989
p.193) but diazo dye like trypan blue may be used without activation (Wang et al.,
20
1997; Chapalamadugu, 2006; Chapalamadugu et al., 2008) . Owen (1985)
compared results of in vitro activation with various vital stains and concluded
that the subjective differences between stained and unstained eggs caused
inconsistent results and that only diazo dyes yielded significant results.
Chapalamadugu et al. (2006; 2008) concluded that T. taeniaeformis non activated
eggs that were heat treated and ex–shelled with 0.5% sodium hypochlorite did
not yield consistent results for staining of oncospheres with various non –vital
(acridine orange) and vital (trypan blue, propidium iodide) dyes.
1.7.3 IN VITRO CULTURE
Heath and Smyth (1970) described in in vitro culture technique for
development of oncospheres of T. hydatigena , T. ovis , T. pisiformis , T. serialis and
Echinococcus granulosus to cystic larvae stages. Despite being a successful
technique, it was not quantitative enough to access viability rates and was limited
to studying protective antigenic response of relevant intermediate host species
and vaccine development (Lightowlers et al. , 2003; Kyngdon et al., 2006).
1.7.4 IN VIVO AND SURROGATE MODELS
In vivo studies may yield the most reliable results provided intermediate
and definitive hosts are available for the target species or appropriate surrogate
species is selected. Due to greater risk of human infe ction and low availability of
T. saginata, working with this tapeworm is a challenging task. To resolve this
issue, a surrogate model has been adopted for this study that involves a
21
phylogenetically very similar tapeworm (T. hydatigena), which has a ruminant
intermediate host (sheep) and a canine definitive host (dog).
Taenia hydatigena is phylogenetically very similar to T. saginata and falls
in the same subclade in a phylogenetic tree of genus Taenia. T. hydatigena is more
phylogenetically similar to T. saginata than T. solium and T. taeniaeformis
(Hoberg et al., 2000; Hoberg, 2006) . The predilection site of these metacestodes
is omentum, mesenteries and liver as compared to muscular tissue in T. saginata .
Usually T. hydatigena does not cause any clinical signs in sheep but upon
ingestion of a large dose of eggs or a whole fresh gravid proglottid sudden death
may occur due to hepatitis cysticercosis resembling acute hepatic fasciolosis
(Radostits et al., 2000 p. 1386).
1.8 OBJECTIVES
To justify economical use of the potato co–product, the life cycle of the
tapeworm needs to be intercepted to prevent cattle from getting infected with T.
saginata. For this study our focus was to determine the viability and
characteristics of eggs with following objectives:
1. To study effect of heat treatments ranging from 22 to 60°C for 5 minutes on
viability of eggs. The study was carried out in vitro and in vivo. These heat
treatments were not severe enough to alter the nutritive value of the
potato co–product.
22
2. To study the effect of ensilation of potato co -product for 0 to 28 days on
viability of T. hydatigena eggs.
23
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32
Table 1.1 Taxonomy of tapeworms of genus Taenia.
Category Taxonomic Classification
Characteristics
Kingdom Animalia -
Phylum Platyhelminthes Flatworms – body is dorso-ventrally
flattened
Class Cestoidea Hermaphrodite, Lack body cavity and
alimentary canal
Sub-class Eucestoda Hexacanth embryo, body of adult divided
into scolex, neck and strobila
Order Cyclophyllidea Two-host life cycle, large tapeworms
Family Taeniidae Poorly developed eggs shell, thick
keratinized embryophore
(Soulsby, 1982; Smyth and McManus, 1989; Singh and Prabhakar, 2002;
Mehlhorn, 2006)
33
Table 1.2 Reported bovine cysticercosis outbreaks in North America.
Year Location Source of Epidemic Reported by
1973 Phoenix,
Arizona
Farm worker (Slonka et al., 1975)
1978 South
California
Farm worker (Slonka et al., 1978)
1981 Ohio Leakage of raw sewage
onto pastures after flood
(Fertig and Dorn, 1985)
1984 Washington Human fecal
contamination of local
feed sources
(Hancock et al., 1989)
1987 West Texas – Not reported – (Weedon, 1987)
1992–93 Idaho Potato co–products (Yoder et al., 1994)
2000 Alberta,
Canada
Leakage of sewage onto
fields
(Lees et al., 2002;
Scandrett and Gajadhar,
2004)
34
Table 1.3 Estimates of economic losses per animal due to cysticercosis .
Location Loss per animal Reference
West Texas $253 (Weedon, 1987)
South–central Idaho $337 (Yoder et al., 1994)
West US states average $1186* (Getz et al., 2008)
* Based on cost of an average 750 lb carcass. These losses do not include losses
due to decrease in reputation; raising, feeding and maintenance cost of the beef
animals.
35
Figure 1.1 Critical control points in life cycle of Taenia saginata.
C D
B A
Infected feces contaminating feed
stuffs
Infected Beef
Cattle
Humans
36
Chapter 2 THERMAL KILLING OF TAENIA HYDATIGENA EGGS
37
Abstract
In the Pacific Northwest USA feeding of potato co–products has been
speculated to result in greater prevalence of beef cysticercosis caused by Taenia
saginata as compared to rest of the USA. A Taenia hydatigena model was used to
assess the effect of heat treatments on viabilities of eggs below temperatures of
complete potato gelatinization. The T. hydatigena life cycle was maintained under
laboratory conditions by passing the parasite through a canine –ovine cycle. The
eggs were used for in vitro and in vivo studies to determine effects of five minutes
of heat treatment, ranging from room temperature (22°C) to 60°C, on viabilities
of the eggs. The results showed 99.47 and 100% reduction in viabilities after five
minutes of heat treatment at 60.00 and 57.38°C under in vitro and in vivo
conditions, respectively. Similar heat treatments m ay also be effective against T.
saginata and may help to reduce occurrence of beef cysticercosis.
Key words: Taenia saginata , Taenia hydatigena , cysticercosis, potato co–product,
heat treatment, potato gelatinization .
2.1 INTRODUCTION
Taenia saginata is a tapeworm of humans that causes cysticercosis in
intermediate hosts (cattle) and taeniosis in definitive hosts (humans). Being a
public health concern, strict post mortem regulations apply at the harvest level
that leads to condemnation of infected carcasses and thus economic losses to beef
producers. Cattle become infected orally; most commonly through contaminated
38
water, feed stuffs or infected farm workers. Depending on the extent of exposure
the disease may be endemic or epidemic in cattle (Slonka et al., 1975; Slonka et
al., 1978; Fertig and Dorn, 1985; Weedon, 1987; Hancock et al., 1989; Yoder et al.,
1994; Lees et al., 2002; Scandrett and Gajadhar, 2004) . Based on unpublished
United States Department of Agriculture (USDA) meat inspection data, in 2006
the prevalence of beef cysticercosis was 0.067% in the Pacific Northwest
compared to 0.004% in the USA. There were 720 and 256 reported cysticercosis
cases in the Pacific Northwest and rest of the USA, respectively. Based on the
same data source, the levels of the disease in the Pacific Northwest have been
consistently higher than the rest of the USA at least since 1984. T. saginata eggs
originating from humans are the infective stage to cattle leading to cysticercosis.
Potato co–products are considered a likely source of T. saginata eggs leading to
elevated levels of disease in the region (Hancock et al., 1989; Yoder et al., 1994) .
Contamination of potato co–products with T. saginata eggs may occur in the field
at potato processing plants or at feedlots. Since the proglottids containing eggs
are motile and can actively migrate out of infected humans, contamination of
potato co–products may not always require direct contact with human feces. To
justify economical use of the potato co –product, intervention in the life cycle of
the tapeworm is needed to prevent cattle from getting infected. One intervention
to prevent transmission to cattle involves heat treatment of the potato co –
product to kill viable T. saginata eggs before cattle ingest them. In this context,
heat treatments were used by some beef operations in the region which were
39
excessive and caused gelatinization of the potato starch. Gelatinization of this
kind caused ruminal acidosis (Nelson, 2003). A possible control strategy under
this situation may be heating the potato co –product to an optimum time–
temperature combination below the initiation temperature for gelatinization of
potato starch that will effectively kill T. saginata eggs. The author is not aware of
any research studies that have made these measurements.
Measurements used by different investigators in in vitro and in vivo studies
to assess viability of Taenia spp. eggs include staining, oncospheres activation
analysis, culture, infection of intermediate hosts and lab animals (Williams and
Colli, 1970; Wang et al., 1997; Minozzo et al., 2002; Chapalamadugu, 2006;
Kyngdon et al., 2006; Chapalamadugu et al., 2008) . Williams and Colli (1970)
studied effect of heat on infectivity of T. hydatigena by inoculating heat treated
ex–shelled and activated oncospheres into jirds (Meriones unguiculatus) but
concluded that the in vivo system was unreliable. Chapalamadugu et al. (2008)
concluded that accurate assessment of viability of T. taeniaeformis eggs using
vital dyes was questionable after heat treatment and ex–shelling with 0.5%
sodium hypochlorite. In vivo studies may yield the most reliable results provided
that the intermediate and definitive hosts are available for the target species or
an appropriate surrogate species is selected.
For this study, viabilities of eggs were evaluated in vitro by measuring
activation rates and in vivo by counting postmortem cysticerci in infected lambs.
40
Due to low availability of T. saginata , T. hydatigena was used as a surrogate
species. T. hydatigena has a close phylogenetic relationship with T. saginata
(Hoberg et al., 2001). A major difference between life cycles of T. hydatigena and
T. saginata is the site where cysticerci local ize in the intermediate host. T.
hydatigena larvae have affinity for the liver through which they migrate to reach
the abdominal cavity (Soulsby, 1982). To a lesser extent, larvae may reach other
tissues such as lungs (Blazek et al., 1985). In contrast, T. saginata cysticerci
localize in skeletal and cardiac muscles. The objective of this study was to
evaluate effect of heat treatments on viability of T. hydatigena eggs.
2.2 MATERIALS AND METHODS
2.2.1 TAENIA HYDATIGENA EGGS
Taenia hydatigena eggs were obtained from Dr. M.W. Lightowlers
(University of Melbourne, Australia) to maintain the life cycle of the parasite
under laboratory condition using ovine–canine cycle. Three recently weaned
lambs were used for each passage in the intermediate ho st. The dose of eggs
necessary to provide an acceptable assessment of treatments were determined in
a preliminary experiment in which 100, 1,000 or 2,000 eggs were administered
orally to lambs in 2 ml phosphate buffered saline (PBS, pH=7.2). No morbidity o r
mortality was observed in any of the groups, with a mean cysticerci recovery rate
of 10% of eggs administered initially. Thus, the egg dose was determined to be
2,000 eggs per lamb administered orally in 2ml PBS. All lambs in the study were
41
maintained on corn–based starter (36% corn for one week), grower (52% corn
for three weeks) and finisher (76% corn for three to five weeks) diets which met
or exceeded NRC requirements (NRC, 2007). Lambs were housed indoors in
temperature controlled room (19–21°C) with 13 hrs of light per day. Lambs were
slaughtered eight weeks after infection and calcified (dead) or non –calcified
(alive) cysticerci were recovered from omentum, liver surface, peritoneal cavity,
diaphragm and lungs. Non–calcified cysticerci were collected in PBS and up to six
cysticerci were orally administered to two dogs within 30 –60 minutes of
collection.
Two dogs were used as the definitive host for the entire study. One dog was
a six year old castrated red–tick coonhound, and the other was a seven year old
castrated blue–tick coonhound. The dogs were fed a commercial adult dog
maintenance diet (2021 Teklad global 21% protein dog diet, Harlan laboratories,
USA) and kept in temperature controlled (19 –21°C) indoor kennels with 13 hrs of
light per day. The dogs began shedding proglottids from seven to nine weeks after
infection with cysticerci from lambs, similar to results of Gregory (1976). The
proglottids shed by dogs were found motile and were recovered from the floor or
occasionally from feces. Up to six proglottids were recovered per dog per day and
were collected in 1% Antibiotic–Antimycotic solution (AA solution, Sigma Chem.,
USA) in PBS and stored at 4°C. Proglottids were not di ssected to collect eggs,
since active contractions of the proglottids expelled most of the eggs out into the
42
solution within few hours of collection. The eggs were stored at 4°C and were
used within one month of collection.
2.2.2 IN VITRO EXPERIMENT
In a completely randomized design, temperature treatment s were carried
out in duplicate at 40, 45, 50, 55 and 60°C for five minutes each. Heat treatments
were selected based on a preliminary experiment in which five minutes of heat
treatments at 40, 50 and 60°C resulted in activation rates of 5.88, 2.38 and 0.00%.
The untreated control was maintained at room temperature of 22°C. Egg
concentrations were determined by counting eggs in 10 µl of the egg solution on a
ceramic ring slide (Ring Microflocculation Slide, Clay Adams, USA) with a cover
slip under a 400× magnification of a light microscope (BH–2, Olympus, USA). Four
thousand eggs were diluted to a final volume of 1.2 ml with PBS in 2.0 ml flat
bottom micro–centrifuge tubes for each treatment. Two heat treatments were
carried out at a time using two heat blocks (VWR, Univar, USA), and temperature
was monitored with a real–time digital scanning thermometer (12 Channel
Scanning Benchtop Thermometer, Digi–Sense, Cole–Parmer Instrument Company,
USA).
Following heat treatment, ex–shelling was carried out to remove the
embryophore, using a sodium hypochlorite technique with modifications
(Lightowlers et al., 1984; Wang et al., 1997; Chapalamadugu et al., 2008) . Sodium
hypochlorite (0.3 ml of 6% NaOCl) was added to the heat treated egg solution to a
43
final concentration of 1.2%. The solution was shaken vigorously using a vortex
mixer (SP Deluxe Mixer – S8220, American Scientific Products, USA) to facilitate
disintegration of keratin blocks. After five minutes, the solution was diluted to 2
ml with PBS to dilute the sodium hypochlorite to 0.9% and then washed thrice by
centrifuging at 1,000×g for 10 minutes (Galaxy 7 Microcentrifuge, VWR
international, USA); each time removing 1.6 ml supernatant and diluting to 2 ml
with PBS. After the last wash, the supernatant was removed to retain a volume of
100 µl of ex–shelled oncosphere suspension.
Sheep bile, collected at time of slaughter of lambs, was used for activation
of oncospheres. Bile was collected from gall bladders using a syringe, pooled and
stored at –20°C in 1.5 ml microcentrifuge tubes. For activation, bile was thawed
in a water bath at 37°C and was then added (100µl) to the solution of ex –shelled
oncospheres (100µl) to final bile concentration of 50%. The solution was
incubated in a water bath (Water bath 183, Precision Scientific Inc., USA) at 37°C
for two hours. Two hours of incubation was selected based on results of a
preliminary study in which maximum observed percent activity of onc ospheres
was observed at two hours of incubation compared to other time periods of 0, 0.5,
1, 4, and 8 hours of incubation. To observe activity, 20 µl of the solution was
transferred on to a ceramic ring slide and a cover slip placed on it. Eggs in each
field were individually observed for movement of hooks and contractions
(activation) under a light microscope (BH–2, Olympus, USA) at 400×
44
magnification. Percentage of oncospheres demonstrating activity was then
determined.
Gallie and Sewell (1970) cited by Wang (1997) reported that increased
levels of carbon dioxide in hatching medium improved activation rates of T.
saginata oncospheres but Ishiwata et al. (1993) found that carbon dioxide
actually reduced activation rates of T. taeniaeformis oncospheres. This effect of
carbon dioxide may be a species specific effect and was unknown for T.
hydatigena . Preliminary experiments in the current study demonstrated no effect
of carbon dioxide on activation rates of T. hydatigena oncospheres and thus
carbon dioxide was not used in subsequent experiments.
Heat treatment and exposure to sodium hypochlorite may lead to decreased
oncosphere recoveries at higher temperatures, as suggested by Chapalamadugu et
al. (2008) with T. taeniaeformis at temperatures above 65°C. Therefore the
recovery percent, based on an initial egg count of 4 ,000, was determined and
compared among treatments.
2.2.3 IN VIVO EXPERIMENT
Heat treatments of eggs were carried out at 50 and 60°C for five minutes
each. Untreated control eggs were maintained at room temperature (22°C).
Following heat treatments, three, recently weaned Suffolk lambs (33.3 ±1.07kg)
per treatment were infected orally , via syringe, with 2,000 heat treated or
untreated control eggs in 2 ml PBS. Lambs were weighted on day 0, 14, 21, 35, 49
45
and 56 post–infections to monitor any effect of treatment on weight gains. Five
and four randomly selected lambs were slaughtered on days 62 and 64 post–
infection, respectively. Captive bolt stunning was used for humane slaughter of
lambs (Welty, 2007; FSIS, 2009). The numbers of cysticerci were counted in
omentum, liver surface, diaphragm, mesenteries, lungs and peritoneal cavity. Bulk
of the liver cysticerci were found on liver surface with occasional cysticerci under
the surface, consequently liver cysticerci counts were restricted to the surface.
The viability was calculated as number of cysticerci recovered, as a percentage, of
heat treated eggs administered.
2.2.4 STATISTICAL ANALYSIS
2.2.4.1 In vitro analysis
Two replicates per treatment were studied in a completely randomized
design (CRD). A sigmoidal four–parameter model (Eqn. 1) (Rutledge, 2004;
Chapalamadugu et al., 2008) was fitted using a non linear regression analysis of
SigmaPlot 11.0 (SPSS Inc, Chicago, USA). Based on the same model, the four
parameters were estimated using Proc NLIN of SAS 9.2 (SAS Institute, Cary,
North–Carolina, USA).
– – – – – [Eqn. 1]
Where, f denotes the dependent variable – percent activity, x denotes
independent variable – temperature treatment, and the four parameters are a
46
(total change in percent activity), b (slope of the curve), x0 (inflection temp) and
y0 (minimum percent activity).
To determine any interactions between heat treatment temperature and
exposure to sodium hypochlorite, the oncosphere recoveries were compared at
different temperatures using analysis of variance (Proc GLM in SAS 9.2).
2.2.4.2 In vivo analysis
Cysticerci recovered as percent of eggs administered (viability) were
regressed on temperature of heat treatment of eggs using linear regression (Proc
REG in SAS 9.2). A split plot design was used to analyze the number of calcified,
non–calcified and total cysticerci recovered from different sites of recovery, with
temperature of heat treatment in the whole plot and its interactions in the sub
plot with individual lamb within temperature as the whole plot error term using
Proc GLM in SAS 9.2. In case a significant temperature × site interaction was
found, slice analysis was carried out within temperature for effect of sites of
recovery on cysticerci count and within recovery site for effect of temperature on
number of cysticerci recovered (Winer, 1971). The weights of lambs on day 0, 14,
21, 35, 49 and 56 post–infection were compared among treatments using analysis
of covariance (ANCOVA) with repeated measures and unequal time intervals
using Proc MIXED of SAS 9.2.
47
2.3 RESULTS
2.3.1 IN VITRO RESULTS
Maximum percent activity was observed at 40°C (6.06 ±0.55%), which
decreased with increased temperature to a point at 60°C when no activity was
observed (Table 2.1). Based on these observations, a sigmoidal four–parameter
model resulted in parameters shown in Table 2.2 (R2 = 0.8718). The data points
and non linear regression model, with 95% confidence and prediction bands, are
shown in Figure 2.1. Based on the non linear model, it was predicted that percent
activity was reduced by 99.47% by heating to at 60°C for five minutes. Total
oncospheres recovered were not significantly different among different
treatments thus indicating no interaction betw een the temperature and exposure
to sodium hypochlorite.
2.3.2 IN VIVO RESULTS
Most of the calcified and non calcified cysticerci were recovered from
omentum and liver surface of lambs infected with eggs treated at 22°C (control)
or 50°C, while no cysticerci were recovered at any site for treatment group dosed
with eggs heat treated at 60°C. Comparative visual postmortem inspection of the
liver surfaces from three treatment groups was found to be indicative of extent of
infectivity across different treatment groups (Figure 2.2).
The mean percentage of cysticerci recovered were 11.55±1.85, 1.22±0.54,
0.00±0.00 % of the total eggs administered for treatments at 22 (control), 50 and
48
60°C, respectively. Linear regression analysis resulted in the equation Y= –0.318
X + 18.249 (R² = 0.877) where Y was the cysticerci recovered as percent of eggs
administered and X was the temperature of heat treatment for five minute s
(Figure 2.3). The slope of the linear equation indicated mortality rate of 0.32% of
total eggs administered per degree Celsius rise in temperature from 22 to 60°C
for five minutes. Additionally, the stat istical model predicted 0% egg viability was
attained at 57.38°C.
Split plot analysis of cysticerci (number of calcified, non–calcified and total
number) recovered showed significant interaction s between the temperature of
heat treatment and the site of recovery (Table 2.3). Subsequent slice analysis
revealed that at 22°C (control) the mean number of cysticerci recovered from
omentum was significantly different from all other sites. However no such
difference was observed at 50°C. Also, the mean number of cysticerci recovered
from omentum, liver, lungs and diaphragm decreased linearly with temperature
of heat treatment but the mean recoveries from mesenteries and peritoneal cavity
for all treatments were too low to predict any relation between temperature and
recovery. The ratio of the calcified to non–calcified cysticerci was found to be
independent of temperature, lamb and site of recovery. The residuals of the count
data of cysticerci recovered from different organs were normally distributed with
skewness of 1.32 and kurtosis of 14.43. This slight deviation from a normal
distribution may be explained by the excessive occurrence of zeros in the count
data, after heat treatments at 50 and 60°C (Table 2.3).
49
The ANCOVA analysis of weights of lambs from different treatment groups
(Table 2.4) indicated no difference in slopes of the curves (weight gain per day)
or intercepts (weight at day 0) among treatments.
2.4 DISCUSSION
In the present study the effects of five minutes of different heat treatments
on the viabilities of T. hydatigena eggs were analyzed in vitro and in vivo. The
most important observations were made at 60°C at which no activation of
oncospheres was observed in vitro and no cysticerci were recovered from lambs
infected with eggs treated at this temperature. Complete potato starch
gelatinization required 30–60 minutes of heat treatment at 64.2°C (Shiotsubo,
1983, 1984). Further, 60 minute treatments of potato starch at 55, 57.5, 60, 62.5
and 65°C resulted in 0, 46, 73, 88 and 100% gelatinization, respectively (Parada
and Aguilera, 2009). Thus, treating potato co–product with temperature and time
treatments sufficient to neutralize infectivity of oncospheres appears to
represent conditions that marginally cause gelatinization of potato starch. It will
be important to more accurately determine optimal temperature and time
treatments that maximize egg killing and keep gelatinization to minimum. Other
experiments indicate that ensilation of potato co –products is detrimental to T.
hydatigena eggs (Buttar et al., 2009). Hence a combination of heat and ensiling
treatments might reduce the intensity of heat required for this purpose.
50
In vitro and in vivo statistical models indicated a 99.47 and 100.00%
reduction in viabilities after five minutes of heat treatment at 60.00°C and
57.38°C, respectively. The slope of the curve indicated mortality rate of
oncospheres in response to increasing temperature was estimated to be 2.25 and
0.32% of total oncospheres per degree Celsius increase in temperature for in vitro
and in vivo models, respectively. Differences in results of in vitro and in vivo
models may be due in part to use of different statistical approaches – non linear
sigmoidal four–parameter model for in vitro experiment and linear regression for
in vivo experiment. Nevertheless, it is also possible that the difference reflects
sensitivity of in vitro assay to detect viable eggs. More data points are
recommended for in vivo experiment between 22°C and 60°C to better fit a non
linear regression model.
When compared to control (22°C), the in vivo cysticerci recovery reduced
to 10 and 0% for 50°C and 60°C treatment groups, res pectively. Similarly, the in
vitro activation rates reduced to 120, 88, 38, 5 and 0% of activation rates at 22°C,
for eggs heat treated at 40, 45, 50, 55 and 60°C, respectively. Similar results were
reported by Williams and Colli (1970) that no in vitro activation of T. hydatigena
oncospheres was observed after heat treatment at 55°C for 5 minute, 60°C for 2
minute and 65°C for 1 minute. The results presented here are also similar to the
predicted thermal death point (56°C for 5 min) of T. saginata cysticerci (Allen,
1947). However, Pike (1988) and Hughes et al. (1985) cited by Bruce et al. (1990)
reported a prolonged heat requirement (55°C for 3 hours) to reduced infectivity
51
of T. saginata eggs to 1% in sludge. This difference may be due to lack of
penetration of heat through the sludge, protecting the eggs for prolonged period.
Similar situation may apply to potato co–products. Percent active oncospheres
recovered for heat treatment at 40°C (5.98±0.55%) were similar to that at 22°C
(4.98±1.36%). This may indicate that heat treatment at 40°C for five minutes has
no effect on viability of T. hydatigena eggs. Moreover, for activation all eggs in the
in vitro study were incubated in bile at 37°C for two hours, which is a thermal
treatment that may be more intense than 40°C for five minutes, thus further
strengthening the possibility that 40°C may not reduce viability of T. hydatigena
eggs.
In vitro exshelling and activation are important steps required to study
viability of Taenia spp. The most widely adopted in vitro approaches have been
exshelling with sodium hypochlorite (Lightowlers et al., 1984; Wang et al., 1997;
Chapalamadugu et al., 2008) and activation with species–specific bile (Ishiwata et
al., 1993; Wang et al., 1997; Kyngdon et al., 2006) . For this study we used 1%
sodium hypochlorite for exshelling and 50% sheep bile for activation in vitro.
Viabilities of T. hydatigena eggs were not assessed using in vitro vital staining in
the current study since accurate assessment of viability with staining after heat
treatment is questionable (Chapalamadugu, 2006; Chapalamadugu et al., 2008) .
Instead activation rates were used to assess viability. Wang et al. (1997) reported
activation rates of 4, 1% and viabilit ies (based on trypan blue staining) of 80,
87% for enzymatic and sodium hypochlorite ex–shelling techniques, respectively.
52
All viable oncospheres may not activate at same time and this may be the reason
for the difference in percent activation rates and st aining observed by Wang et al.
(1997). Different approaches have been suggested by m any investigators to
improve activation rates but methods that yield consistent results remain
unresolved. In the results presented here, in vitro activation rates were found
consistent with maximum activation rate of 6% after two hours of incubation in
bile. Activation of all viable oncospheres may not have been observed but
comparison of results among treatments and comparison to in vivo results
strengthens the in vitro technique used for this study.
Dead cysticerci in an intermediate host often calcify in response to the
host’s immune system. The ratio of calcified to non –calcified mainly depends on
host’s age, immune response and number of days since infection (Minozzo et al.,
2002; Mehlhorn, 2006). Since the lambs used in the study were of same age and
were slaughtered only two days apart, the ratio of calcified to non–calcified cysts
did not vary significantly. Also, there was no effect of temperature of treatment of
eggs and site on calcification status of cysticerci.
No morbidity or mortality was observed in the lambs used in in vivo
experiment. The rates of weight gain did not vary significantly among treatments
even though cysticerci recovered did differ significantly. These were expected
observations since T. hydatigena does not cause any clinical signs in sheep
(Soulsby, 1982).
53
The results reported here present a promising foundation for application of
mild heat treatments that effectively kill T. saginata eggs without causing
significant starch gelatinization of potato co–products. Further studies should be
conducted to better understand the effect of various time–temperature
combinations of heat treatments without potato gelatinization. This may help to
understand the relationships of time and temperature combinations on egg
viabilities of Taenia spp. eggs. The thermal treatments may be investigated
further by using T. saginata instead of surrogate species. Such studies may help in
predicting more time–temperature combinations for practical application at level
of potato processing plants to render potato co–products free of infectious T.
saginata eggs.
Lagooning of sludge at 7°C for 28 days caused more than a 99% reduction
in viability of T. saginata eggs (Bruce et al., 1990). A similar approach involving
ensilation of potato co–products may prove beneficial to control T. saginata and
needs to be explored further. Additionally, analysis of current epidemiological
data of beef cysticercosis in the Pacific Northwest, similar to Yoder et al. (1994)
and Hancock et al.(1989), will help better understand the current disease
dynamics in the region.
54
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59
Table 2.1 In vitro effect of five minutes of thermal treatment of T. hydatigena
eggs on percent recovery of oncospheres and percent activation ex–shelled
with 1% sodium hypochlorite and activated with 50 % bile treatments.
Temperature (°C) Recovery ± SE (%) a Activation ± SE (%) b
22 3.76 ± 0.46 4.98 ± 0.96
40 3.07 ± 1.02 5.98 ± 0.31
45 3.34 ± 0.74 4.87 ± 1.63
50 2.37 ± 0.45 2.11 ± 0.34
55 3.84 ± 0.66 0.33 ± 0.28
60 1.51 ± 0.33 0.00 ± 0.00
a The percent recovery was calculated based on total egg count of subsample
compared to initial count of eggs used.
b The percent activation is based on number of oncospheres found active out
of total number of eggs counted in subsample. The effect of temperature
was non–significant on the percent recovery (p=0.2139) and significant on
percent activation (p<0.0001).
60
Table 2.2 Sigmoidal four–parameter model parameter estimates for in vitro
percent activation of ex–shelled T. hydatigena eggs in response to heat
treatment for five minutes at various temperatures.
Parameter Estimate Standard Error 95% Confidence Limits
a 5.700 0.799 3.942 7.459
b –2.247 1.101 –4.670 0.177
x0 48.666 1.147 46.142 51.190
y0 –0.028 0.534 –1.203 1.146
a = Total change in percent activity (%)
b = Slope of the curve (% /°C)
x0 = Inflection temperature (°C)
y0 = Minimum percent activity (%)
Table 2.3 Average number of calcified and non-calcified cysticerci recovered from lambs infected
with T. hydatigena eggs heat treated for five minutes at 22 (control), 50, 60°C .
Temperature of heat treatment for 5 minutes
22°C (Control)
50°C
60°C
Organ Calcified Non-
Calcified Total Calcified Non-
Calcified Total Calcified Non-
Calcified Total
SE =12.86 SE =8.08 SE =17.04 SE =2.91 SE =0.96 SE =3.56 SE =0.00 SE =0.00 SE =0.00
Omentum* 124.0 a 54.3 a 178.3 a 10.0 2.7 12.7 0.0 0.0 0.0
Liver* 25.3 b 10.7 b 36.0 b 9.3 2.0 11.3 0.0 0.0 0.0
Diaphragm* 5.7 b 2.7 b 8.3 b 0.0 0.0 0.0 0.0 0.0 0.0
Lungs* 5.0 b 1.0 b 6.0 b 0.0 0.3 0.3 0.0 0.0 0.0
Mesenteries 0.7 b 0.3 b 1.0 b 0.0 0.0 0.0 0.0 0.0 0.0
PC 0.3 b 1.0 b 1.3 b 0.0 0.0 0.0 0.0 0.0 0.0
Within a column different superscripts indicate difference among sites of recovery (p<0.05)
* Increasing temperature had linear effect on total recover (p<0.05)
SE = Standard error
PC = Peritoneal cavity
61
Table 2.4 Average weight (±SE) of lambs infected with heat treated T. hydatigena eggs at different
temperatures. The effect of heat treatment on weight gains was not significant.
Treatment Group Day 0 (kg) Day 14 (kg) Day 21 (kg) Day 35 (kg) Day 49 (kg) Day 56 (kg)
22°C (Control) 34.02 ± 1.13 35.49 ± 1.25 37.99 ± 1.93 43.54 ± 3.63 48.53 ± 4.31 51.71 ± 4.99
50°C 31.9 ± 2.69 33.87 ± 2.56 36.29 ± 2.75 39.76 ± 3.27 43.85 ± 3.94 46.87 ± 4.58
60°C 34.17 ± 1.71 37.19 ± 1.49 39.61 ± 1.88 45.96 ± 2.55 51.03 ± 3.32 54.81 ± 4.06
62
63
Figure 2.1 Sigmoidal four–parameter model for observed In vitro percent
activation of T. hydatigena oncospheres after heat treatment for five minutes at
various temperatures (R2 = 0.8718).
Temperature (°C)
20 30 40 50 60 70
Pe
rce
nt
activa
tio
n (
%)
-4
-2
0
2
4
6
8
10
95% Confidence Band
95% Prediction Band
64
a
b
c
Figure 2.2 Comparison of liver surfaces of lambs infected with T. hydatigena
eggs heat treated at (a) 22 (control), (b) 50 and (c) 60°C.
65
Figure 2.3. Linear regression response of percent recovery of T. hydatigena
cysticerci recovered as a percent of heat treated eggs administered at
various temperatures. (Y= –0. 318 X + 18.249, R² = 0.8767)
0%
2%
4%
6%
8%
10%
12%
14%
16%
18%
20%
0 10 20 30 40 50 60 70
Cy
sts
reco
ve
red
as
pe
rce
nt
of
eg
gs
ad
min
iste
red
(%
)
Temperature (°C)
66
Chapter 3 EFFECT OF ENSILATION OF POTATO ON VIABILITY OF TAENIA
HYDATIGENA EGGS
67
Abstract
A Taenia hydatigena model was used to assess the effect 0, 7, 14, 21, and 28
days of ensilation of minced potato on viability of tapeworm eggs. For infection of
lambs, 2,000 T. hydatigena eggs were ensiled in minced potato at room
temperature and fed to recently weaned lambs (29.9±0.76kg). At slaughter after
49 days, no cysticerci were recovered from lambs infected with eggs ensiled for
28 days while a mean of 5.0±5.0 cysticerci (0.25% of the initial egg dose) were
recovered from lambs infected with eggs ensiled for 21 days . For lambs fed eggs
ensiled for 0 days (control), 359.3±55.6 cysticerci were recovered (18.0% of the
initial egg dose). Regression analysis revealed that maximum reduction in
viability was attained after 18.59 days of ensilation.
Key words: Taenia saginata , Taenia hydatigena , cysticercosis, potato co–product,
ensilation.
3.1 INTRODUCTION
Taenia saginata is a worldwide prevalent cestode, with humans as
definitive host and cattle as intermediate host. Cattle are infected by ingesting
eggs shed by humans. Various factors that may contribute to transmission of eggs
from humans to cattle include poor sewage disposal (Cabaret et al., 2002); poor
hygienic practices of farm workers (Slonka et al., 1975; Slonka et al., 1978;
Murrell and Dorny, 2005); water contamination (Fertig and Dorn, 1985); and
transmission of eggs by birds, arthropods and earthworms (Murrell and Dorny,
2005). Based on USDA data, the Pacific Northwest USA has a relatively high
68
prevalence rate of beef cysticercosis in finishing cattle compared to the rest of
USA. This high prevalence was attributed to widespread feeding of potato co –
products in the region (Hancock et al., 1989; Yoder et al., 1994) . Potato co–
products have excellent feeding value (Nelson et al., 2000), no detrimental effect
on meat quality (Busboom et al., 2000) and is readily available available in the
Pacific Northwest. Thus potato co–products present an economic feeding option
for the feedlots and also helps to dispose of the co–product generated by the
potato industry in an efficacious and profitable way (Nelson, 2009). Considering
the economic justification of use of the potato co–products in the region it is
important to control the problem of beef cysticercosis by preventing
contamination or treating potato co–products. Since it is not easy to determine
the exact origin of contamination of potato co–products with T. saginata eggs,
treatments to kill T. saginata eggs in potato co–products may be necessary steps
in eliminating beef cysticercosis.
The survivability of Taenia spp. eggs in the environment is very dependent
on environmental conditions and has been reviewed by Murrell and Dorny
(2005). While the survivability of Taenia spp. eggs may be more than 400 days on
pastures (Duthy and Someren, 1948), feed treatments like drying and ensilation
reduce their survivability. The survivability is reduced to 21 days in stored dry
hay (Lucker and Douvres, 1960; Murrell and Dorny, 2005) and 60–80 days in
grass silage at 10°C in Germany (Enigk et al., 1969). By lagooning at 7°C for 28
69
days more than a 99% reduction in infectivity of T. saginata was reported (Bruce
et al., 1990).
Potato co–products have high moisture contents that are not easy to
dewater without losing nutritive value. Additionally, moderate to low
environmental temperature in the Pacific Northwest favors survivability of T.
saginata eggs in wet feedstuffs like potato co–products. Potato co–products have
been ensiled for at least 21 days in different experiments at Washington State
University, and in no case has cysticercosis has ever been reported from cattle fed
these products. In contrast higher prevalence of cysticercosis at other feedlot
operations in the region that use similar potato co–products but do not ensile for
that long (Nelson, 2009). Thus ensilation treatment of potato co –products may
provide a pre feeding treatment that is effective in reducing transmission of T.
saginata to cattle in potato co–products.
The objective of this study was to determine the effect of ensilation of
potato on viability of Taenia spp. eggs using a surrogate species T. hydatigena , a
canine–ovine tapeworm that has a close phylogenetic relationship to T. saginata
(Hoberg et al., 2001; Hoberg, 2006). T. hydatigena was used here because of the
unavailability of T. saginata eggs which are obtained from humans with an adult
tapeworm. A major difference in the life cycle of the two species is the site of
recovery of the cysticerci in the intermediate host – muscle tissue for T. saginata
and the abdominal cavity for T. hydatigena (Soulsby, 1982) making T. hydatigena
cysticerci easier to find and quantify . Therefore, T. hydatigena eggs were ensiled
70
for different lengths of time with potato co –products and fed to lambs to assess
the change in viability of T. hydatigena eggs over length of ensilation.
3.2 MATERIALS AND METHODS
3.2.1 EGGS
Taenia hydatigena eggs were obtained from Dr. M. W. Lightowlers
(University of Melbourne, Australia). Life cycle of the parasite was maintained
under laboratory condition by passing parasite stages between lambs and dogs
and eggs were recovered from dogs as described by Bu ttar et al. (2009). A fresh
batch of eggs was used for each treatment. All the batches came from a single dog
in same infection period. While it may be argued that different batches of eggs
might have variation in viability, the method used here ensured that there was no
variation due to prolonged storage and likely resembled the natural process of
contamination of potato co–products.
3.2.2 ENSILATION
A completely randomized design was used to compare four ensilation
treatments and a non–ensiled control with three replicates. Treatments involved
ensilation of T. hydatigena eggs in minced potato for 0, 7, 14, 21 and 28 days.
Fresh eggs, collected within a week from an infected dog, were used for each
treatment. On day 0, 7, 14 and 21, 2,000 eggs were transferred to freshly minced
red potatoes in each of three labeled, snap–top 30ml plastic vials with a sealable
lid (Olympia Plastics, Los Angles, USA) for ensilation treatments of 28, 21, 14 and
7 days, respectively. The containers were flushed with CO 2 and sealed to promote
71
ensilation. To ensure proper ensilation pH measurements were taken on day 0
and day 5. The containers were immersed into a 5L plastic bucket with a lid
containing the stock of minced potato and then sealed at room temperature.
Three replicate were produced for each length of ensilation. On day 28, contents
were emptied from vials, separately mixed with 0.5 kg corn and i ndividually fed
to one of the 15 different randomly selected, recently weaned lambs
(29.9±0.76kg) off feed for 24 hours but provided with water ad libitum.
3.2.3 LAMBS
Lambs were fed a corn–based starter (36% corn for one week), a grower
(52% corn for three weeks) and a finisher (76% corn for four weeks) diets which
met or exceeded NRC requirements (NRC, 2007). Throughout the study, lambs
were housed indoors in a temperature controlled room (19 –21°C) with 13 hours
of light per day. Lambs were weighed on day 0, 13, 27, 36 and 49. Eight lambs
were randomly selected for harvest at WSU Meats laboratory on day 49 and seven
on day 50 post infection. Captive bolt stunning was used for humane slaughter of
lambs (Welty, 2007; FSIS, 2009). At slaughter, calcified (dead) and non–calcified
(alive) cysticerci were recovered from omentum, liver surface, diaphragm, lung s,
mesenteries, peritoneal cavity, visceral surfaces of rectum, small intestine and
large intestine of each carcass. Percent viability of eggs was calculated for each
lamb by comparing total number of cysticerci recovered to number of eggs
(2,000) initially administered.
72
3.2.4 STATISTICAL ANALYSIS
Cysticerci recovered, as percent of eggs dosed (viability), were regressed
against days of ensilation of eggs in potato co–products using a linear plateau
model with Proc NLIN of SAS 9.2 (SAS institute, Cary, North –Carolina, USA). The
equation used for the regression was Y = β X + i where Y was dependent variable
(percent cysticerci recovered), X was the independent variable (days of
ensilation), β was slope of the non-plateau curve (percent change in number of
cysticerci recovered per day) and i was Y intercept (percent cysticerci recovered
at zero days of ensilation). The inflection point in the model, where the plateau
started was also iterated by Proc NLIN of SAS 9.2.
Assuming a normal distribution, a split plot design was used to analyze the
number of calcified, non-calcified and total cysticerci recovered from different
sites, with days of ensilation in the whole plot and site of recovery and its
interactions in the subplot using Proc GLM Proc GLM of SAS 9.2. Lamb within
temperature was the whole plot error term. In case a significant days of
ensilation × site of recovery interaction was found, data were analyzed to
compare recoveries from different sites, within each treatment, using slice
analysis along with Tukey’s multiple comparison test for means in Proc GLM of
SAS 9.2 (Winer, 1971). The average daily weight gains of lambs were compared
among treatments using Proc GLM of SAS 9.2.
73
3.3 RESULTS
The pH of the minced potato dropped from 6.38 on day 0 to 4.81 on day 5,
ensuring proper ensilation (Nelson, 2009). Larval stages were recovered as
calcified (dead) or non–calcified (alive) cysticerci from omentum (45%), liver
surface (21%) and other sites from all treatments except when ensiled for 28
days (Table 3.1). The cysticerci recovered as a percent of initial dose of 2,000
eggs ensiled, decreased with time of ensilation for 0, 7, 14, 21 and 28; which
averaged 17.97±2.78%, 13.15±4.19%, 3.97±2.21%, 0.25±0.25% and 0.00±0.00%,
respectively. Non–linear regression analysis indicated that cysticerci recovery
decreased at a rate of 1.00±0.24 % per day (slope of curve) with 18.69±2.19%
eggs initially viable at day 0 (intercept). The plateau of the model was reached at
18.59±3.65 days of ensilation at which the percent recovery rate was 0.10±3.72 %
(Figure 3.1). Nevertheless, a 28 day treatment produced no cysticerci.
Split plot analysis of number of calcified, non–calcified and total cysticerci
recovered resulted in a significant interaction between days of ensilation and site
of recovery. Subsequently, slice analysis showed that the means for total recov ery
of cysticerci from omentum and liver of control lambs were significantly different
from all other sites. For lambs with eggs ensiled for 7 days, total mean recovery
of cysticerci from the omentum was significantly different from all other sites,
except liver (Table 3.1). For lambs fed eggs from all other treatments (14, 21 and
28 days) the mean recoveries were not significantly different among differ ent
sites. The residuals of the count data of cysticerci recovered from different organs
74
had skewness of 1.19 and kurtosis of 6.74. This slight deviation from the normal
distribution may be explained by the excessive occurrence of zeros in the count
data, especially from the longest ensilation treatments (Table 3.1).
Lambs in all treatment groups and control gained significant weight post
infection till day of slaughter; and there was no effect of ensilation treatment
time on average daily weight gains of lambs (Table 3.2).
3.4 DISCUSSION
The present study was conducted to determine the effect of 0, 7, 14, 21, and
28 days of ensilation of potato co–products on in vivo viabilities of T. hydatigena
eggs. The most striking result was when no cysticerci were recovered, after 28
days of ensilation of Taenia spp. eggs in minced potato indicating 100% reduction
in viability of T. hydatigena eggs by 28 days of ensilation. These results are
similar to the effect of lagooning on sludge at 7°C for 28 days, which caused more
than a 99% reduction in viability of T. saginata eggs (Bruce et al., 1990). Also in
the present study, the percent recovery after 21 days of ensilation was
0.25±0.25% indicating a substantial decrease in viability by day 21. Further, the
linear plateau model analysis showed that maximum reduction in viability was
reached by 18.59±3.65 days with the reduction in viability at the rate of
1.00±0.24 % per day.
The recovery of cysticerci was mostly from omentum and liver similar to
that reported in a previous study (Buttar et al., 2009). However the percent
75
recovery of cysticerci of control treatments with an initial dose of 2 ,000 eggs was
different for the two studies. In the current study, T. hydatigena eggs were
administered by feeding contaminated minced potato mixed with c orn. This
method resulted in recovery of a maximum of 19% of the dose as cysticerci in
control lambs compared to recovery of 12% in a previous study in which 2,000 T.
hydatigena eggs were administered orally to lambs in 2 ml phosphate buffered
saline. Although minimal, this difference may be due to variable infective
potential of different batches of eggs, different methods of administration or
differences in susceptibility of lambs. Despite the high rate of infection in day 0
and 7 lambs, average daily weight gain was not impacted by treatment (p>0.05).
The survivability of Taenia spp. eggs is highly dependent on environmental
conditions (Murrell and Dorny, 2005) but even under similar conditions the
survivability of different batches of eggs may be quite variable (Froyd, 1962).
High and low temperatures have a detrimental effect on survivability of Taenia
spp. eggs in the environment. Under laboratory conditions, mild temperatures in
range 7 to 38°C promote maturation of juvenile eggs and degeneration of mature
eggs whereas at –9°C, the maturation of juveniles is limited but survival time
does not increase (Gemmell, 1977). Various ensilation treatments in the present
study were carried out at room temperature (22°C). Ensilation in the field with
different temperatures may change survivability of Taenia spp. eggs and needs to
be explored further.
76
Effects of higher temperatures were studied by Buttar et al. (2009), in
which heating T. hydatigena eggs for five minutes at 60.6°C and 57.4°C rendered
them completely unviable based on in vitro and in vivo assays, respectively. The
detrimental effect of ensilation at feedlots, when coupled with heat treatment,
may indicate both milder heat and shorter ensilation times to effectively kill
Taenia spp. eggs. Because ensilation is a natural process and less resource
intensive, this hypothesis should be explored further.
The results obtained with T. hydatigena eggs identify two complementary
treatments that have potential for practical application because , although the
experiments were not conducted on T. saginata, the results are likely to control
beef cysticercosis in feedlot cattle in which potato co–products are fed. It will be
difficult to conduct similar experiments on T. saginata without reliable source of
eggs. Nevertheless, treatment parameters defined here should make analysis
much more efficient with any related experiments on T. saginata when they
become available. In addition, the effect these treatment methods have on T.
saginata transmission to cattle in the field can be monitored by using
epidemiological methods described by Yoder et al. (1994) and Hancock et al.
(1989). Analysis of current epidemiological data in the Pacific Northwest will
help better understand the current disease dynamics in the region.
77
3.5 REFERENCES
Bruce, A.M., Pike, E.B., Fisher, W.J. 1990. A Review of Treatment Process Options
to Meet the EC Sludge Directive, pp. 1-13.
Busboom, J.R., Nelson, M.L., Jeremiah, L.E., Duckett, S.K., Cronrath, J.D., Falen, L.,
Kuber, P.S., 2000, Effects of graded levels of potato by-products in barley-
and corn-based beef feedlot diets: II. Palatability. J Anim Sci 78, 1837-1844.
Buttar, B.S., Nelson, M.L., Busboom, J.R., Jasmer, D.P., Hancock, D.D., Walsh, D.,
2009, In vitro analysis of effect of time–temperature combinations on
viability of Taenia hydatigena eggs. J. Anim. Sci. 87, E-Suppl. 2 / J. Dairy Sci.
92, E-Suppl. 1, 57.
Cabaret, J., Geerts, S., Madeline, M., Ballandonne, C., Barbier, D., 2002, The use of
urban sewage sludge on pastures: the cysticercosis threat. Veterinary
research 33, 575-597.
Duthy, B., Someren, V.V., 1948, The survival of Taenia saginata eggs on open
pasture. East African Agricultural and Forestry Journal 13, 147-148.
Enigk, V.K., Stoye, M., Zimmer, E., 1969, [Die Lebensdauer von Taenieneiern in
Gärfutter]. Deutsche Tierärztliche Wochenschrift 76, 421-444.
Fertig, D.L., Dorn, C.R., 1985, Taenia saginata cysticercosis in an Ohio cattle
feeding operation. Journal of the American Veterinary Medic al Association
186, 1281-1285.
Froyd, G., 1962, Longevity of Taenia saginata eggs. The Journal of parasitology 48,
279.
78
FSIS 2009. Humane Slaughter of Livestock. In Code of Federal Regulations,
9CFR313.15, Food Safety and Inspection Service, D.O.A., ed. (N atl Archiv.
US, Washington D.C., USA).
Gemmell, M.A., 1977, Taeniidae: Modification to the life span of the egg and the
regulation of tapeworm populations. Experimental Parasitology 41, 314-
328.
Hancock, D.D., Wikse, S.E., Lichtenwalner, A.B., Wescott, R.B ., Gay, C.C., 1989,
Distribution of bovine cysticercosis in Washington. American journal of
veterinary research 50, 564-570.
Hoberg, E.P., 2006, Phylogeny of Taenia: Species definitions and origins of human
parasites. Parasitology international 55 Suppl, S23-30.
Hoberg, E.P., Alkire, N.L., de Queiroz, A., Jones, A., 2001, Out of Africa: origins of
the Taenia tapeworms in humans. Proceedings 268, 781-787.
Lucker, J.T., Douvres, F.W., 1960, Survival of Taenia saginata eggs on stored hay.
Proc. Helminthol. Soc. Wash., D.C. 27, 110-111.
Murrell, K.D., Dorny, P., 2005, WHO/FAO/OIE Guidelines for the Surveillance,
Prevention and Control of Taeniosis/Cysticercosis. OIE (World
Organisation for Animal Health) : WHO (World Health Organization) : FAO
(Food and Agriculture Organization), Paris.
Nelson, M.L., 2009, Utilization and Application of Wet Potato Processing Co -
Products for Finishing Cattle. J. Anim Sci., jas.2009-2502.
Nelson, M.L., Busboom, J.R., Cronrath, J.D., Falen, L., Blankenbaker, A., 2000,
Effects of graded levels of potato by-products in barley- and corn-based
79
beef feedlot diets: I. Feedlot performance, carcass traits, meat composition,
and appearance. J Anim Sci 78, 1829-1836.
NRC, 2007, Nutrient requirements of small ruminants : sheep, goats, cervids, and
New World camelids. National Academies Press, Washington, D.C.
Slonka, G.F., Matulich, W., Morphet, E., Miller, C.W., Bayer, E.V., 1978, An outbreak
of bovine cysticercosis in California. American Journal of Tropical Medicine
and Hygiene 27, 101-105.
Slonka, G.F., Moulthrop, J.I., Dewhirst, L.W., Hotchkiss, P.M., Vallaza, B., Schultz,
M.G., 1975, An epizootic of bovine cysticercosis. Journal of the American
Veterinary Medical Association 166, 678-681.
Soulsby, E.J.L., 1982, Helminths, arthropods, and protozoa of domesticated
animals. Lea & Febiger, Philadelphia.
Welty, J., 2007, Humane slaughter laws. Law and contemporary problems. 70, 175.
Winer, B.J., 1971, Statistical principles in experimental design. McGraw -Hill, New
York.
Yoder, D.R., Ebel, E.D., Hancock, D.D., Combs, B.A., 1994, Epidemiologic findings
from an outbreak of cysticercosis in feedlot cattle. Journal of the American
Veterinary Medical Association 205, 45-50.
Table 3.1 Average number of calcified and non-calcified cysticerci recovered from lambs infected with T.
hydatigena eggs ensiled in minced potato for different time periods.
Days of ensilation
0 (control)
7
14
21
Site of recovery Cal N.Cal Total Cal N.Cal Total N.Cal N.Cal Total Cal N.Cal Total
SE= 9.00
SE = 7.58
SE= 10.67
SE= 10.61
SE= 9.69
SE= 15.49
SE= 2.67
SE= 6.78
SE= 7.66
SE= 0.16
SE= 0.76
SE= 0.84
Omentum 38.33 a 119.67 a 158.00 a 44.33 76.00 a 120.33 a 9.00 25.33 34.33 7.00 2.00 9.00
Liver 79.00 b 37.67 b 116.67 a 25.67 21.00 b 46.67 ab 10.67 10.33 21.00 5.33 1.33 6.67
Lungs 12.67 a 0.67 b 13.33 b 11.67 0.00 b 11.67 b 6.00 0.67 6.67 0.00 0.33 0.33
DI 19.00 a 7.33 b 26.33 b 30.67 7.00 b 37.67 b 1.67 4.67 6.33 0.00 0.00 0.00
Rectum 11.67 a 12.33 b 24.00 b 12.67 11.67 b 24.33 b 1.33 1.00 2.33 0.00 1.00 1.00
ME 3.33 a 14.33 b 17.67 b 12.67 5.00 b 17.67 b 0.33 0.00 0.33 0.00 0.00 0.00
PC 1.67 a 1.67 b 3.33 b 0.67 3.00 b 3.67 b 0.67 2.00 2.67 0.00 0.33 0.33
SI 0.00 a 0.00 b 0.00 b 0.00 1.00 b 1.00 b 1.00 0.33 1.33 0.00 0.00 0.00
LI 0.00 a 0.00 b 0.00 b 0.00 0.00 b 0.00 b 3.33 0.00 3.33 0.00 0.00 0.00
Bile Duct 0.00 a 0.00 b 0.00 b 0.00 0.00 b 0.00 b 0.67 0.33 1.00 0.00 0.33 0.33
After 28 days of ensilation recovery from all organs was zero.
Different superscripts within same column indicate significant difference (α=0.05).
Cal: Calcified, N.Cal: Non Calcified SE: Standard Error DI: Diaphragm
ME: Mesenteries PC: Peritoneal Cavity SI: Small Intestine LI: large Intestine
80
Table 3.2 Average weight (±SE) of lambs infected with T. hydatigena eggs ensiled for various time
periods. Average daily weight gains of lambs did not vary significantly with length of ensilation
(α=0.05).
Ensilation length Day 0 (kg) Day 13 (kg) Day 27 (kg) Day 36 (kg) Day 49 (kg)
0 days (control) 32.50 ± 1.17 37.27 ± 2.45 40.53 ± 1.97 45.00 ± 1.85 46.06 ± 1.9
7 days 31.74 ± 1.35 36.14 ± 1.27 39.02 ± 2.14 42.42 ± 1.84 43.64 ± 2.06
14 days 30.53 ± 0.46 34.62 ± 0.40 34.47 ± 0.72 38.64 ± 0.69 40.53 ± 0.67
21 days 29.02 ± 1.22 32.65 ± 1.35 33.94 ± 1.65 37.2 ± 1.51 38.94 ± 1.19
28 days 25.68 ± 1.14 29.55 ± 1.14 30.23 ± 1.94 34.32 ± 1.96 38.71 ± 1.93
81
Figure 3.1 In vivo response of percent recovery of T. hydatigena cysticerci recovered as a percent
eggs administered after ensilation in minced potato for 0, 7, 14, 21 and 28 days. The data points are
average for the treatment with standard error bars. The regression equation was Y= –1.00 X + 18.69
with plateau starting at 18.59±3.65 days.
0
5
10
15
20
25
0 7 14 21 28
Per
cen
t cy
stic
erc
irec
ove
red
Days of Ensilation
82
83
Chapter 4 CONCLUSION
84
Beef cysticercosis, caused by intermediate life stages of T. saginata, has
relatively high prevalence in the Pacific Northwest USA probably due to
widespread feeding of potato co–products. To control the disease one of the
alternatives is to adequately treat potato co–products before feeding at feedlots.
In the present study the efficacies of heat treatment and ensilation of potato were
studied as control measures to reduce the viabilities of Taenia spp. eggs. A T.
hydatigena model was used as a surrogate species and life cycle was maintained
under laboratory conditions.
Five minutes of heat treatment of T. hydatigena eggs at 60°C resulted in
100% reduction of viabilities of eggs both in vitro and in vivo. Similar heat
treatment of potato co–products may significantly reduce or completely eliminate
the threat of T. saginata without causing significant gelatinization of potato
starch. Statistical analysis of the data revealed 99.47 and 100.00% reduction was
obtained after five minutes of heat treatment at 60.00 and 57.38°C under in vitro
and in vivo conditions, respectively. At the industry level, heat treatment of
potato co–products is possible at potato processing plants just before
transportation to feedlots. Heat treated potato co –products may be immediately
transferred to transport trucks where they may retain heat for a sufficient time
period to significantly reduce the number of viable T. saginata eggs.
Exposure of T. hydatigena eggs to 28 days of ensilation in potatoes caused
100% reduced in viability of the eggs. While ensilation is less resource intensive
than heat treatment, it requires much longer time to effe ctively eliminate the T.
85
saginata threat from potato co–products. The ensiling process may be carried out
at feedlots provided that large pits are available for prolonged ensilation. Since
environmental conditions also play role in survivability of T. saginata eggs, the
survivability of the eggs in ensiled potato co–products may vary significantly with
season and region and may be explored further.
Both pasteurization and ensilation may have application at the industry
level as techniques to effectively control beef cysticercosis. Epidemiologically, it
will be interesting to observe the changes in prevalence rates of beef
cysticercosis after application of either or a combination of the two
complementary treatments mentioned in the study at industry level. It is not
known, however, if a combination of the two treatme nts may require milder heat
and/or shorter ensilation times to effectively eliminate T. saginata eggs.
